1. Field of the Invention
The present invention relates to an enhanced chemiluminescent reaction especially for use in a diagnostic assay, particularly immunoassay, and to a diagnostic kit for use in the assay. A chemiluminescent reaction is a chemical reaction which results in the emission of light. The luminescent emission is generally of sufficient duration to enable the light emitted to be detected or measured, and thereby to allow the detection or quantification of an analyte. The chemiluminescent reaction with which this invention is concerned is that between a 2,3-dihydro-1,4-phthalazinedione (DPD), especially luminol, with an oxidant, especially hydrogen peroxide, and a peroxidase enzyme, especially horseradish peroxidase, which catalyses the oxidation of the DPD by the oxidant. The oxidation is accompanied by emission of light.
2. Description of the Prior Art
Luminescent assays making use of the above-mentioned peroxidase-catalysed oxidation of a DPD include three major types. a. Assays wherein horseradish peroxidase is conjugated to a ligand in order to label it and a luminescent reaction is used to detect or quantitate the label. This category includes peroxidase ELISAs. b. Assays wherein luminescent reactions are used to determine free oxidant, peroxidase or luminol. An example of this type of assay is the determination of antibody-linked glucose oxidase by reacting the enzyme/antibody reagent with glucose to form hydrogen peroxide and then measuring the amount of hydrogen peroxide produced by adding luminol and a peroxidase catalyst under controlled conditions to initiate a luminescent reaction. c. Assays wherein a chemiluminescent compound is used directly to label ligands such as proteins, hormones, haptens, steroids, nucleic acids, metabolites, antigens and/or antibodies. The chemiluminescent DPD such as luminol or isoluminol is normally conjugated to a ligand. Chemiluminescence can be detected by adding peroxidase and an oxidant to the reacted conjugate.
A review of luminescent assays has been published by T. P. Whitehead et al., Clinical Chemistry 25, 1531-1546 (1979).
The sensitivity of the peroxidase-catalysed chemiluminescent oxidation of DPDs can be enhanced by including in the reagents an enhancer, namely a 6-hydroxybenzothiazole (European Patent 87959B), a phenol selected from a narrowly defined class (European Patent 116454B or U.S. Pat. No. 4,598,044) or an aromatic amine selected from a narrowly defined class (UK Patent Application 2162946A or U.S. Pat. No. 4,729,950). These patents and the patent application are owned by National Research Development Corporation. European Patent Application Publication No. 219352A (Minnesota Mining and Mfg. Co.) describes various aromatic amines, including some of those previously mentioned in UK Application 2162946A, as enhancers. The best of all these enhancers are believed to be certain of the narrowly defined phenol compounds. When the phenol enhancer patent applications were originally filed, the main claim defined the phenolic compounds by a general formula and by the qualification that they must act as enhancers. However, some compounds within the general formula did not confer significant enhancement. The general formula was accordingly replaced in the granted patents by a narrower definition based fairly closely on 26 named phenolic compounds found to be enhancers. The preferred enhancer is para-iodophenol (PIP). Three others gave significantly better enhancement than the rest. The phenolic enhancers all give a high signal (light output) and a low signal: background ratio (ratio of light output in the presence and absence of peroxidase enzyme), but their characteristics differ in detail. Accordingly, it is an object of the invention to extend the range of effective enhancers. This is a difficult task because no theory or mechanism has been published to explain how one should attempt to select candidate compounds to try as enhancers. It appears that para-substitution by a substituent which is not strongly electron-withdrawing is favoured, but this generalisation is of limited value, leaving thousands of compounds from which to choose.
Additional prior art is referred to below in a separate section following the "Summary of the Invention" without which its context would be unclear.